NEW YORK ─ The Yale School of Public Health is preparing a submission for Emergency Use Authorization from the US Food and Drug Administration for an RNA extraction-free saliva assay that can be used as an alternative to nasopharyngeal swabs.
Saliva is less invasive than nasopharyngeal swabs and has the potential to enable greater consistency in test results, said Anne Wyllie, an associate research scientist at the Yale School of Public Health, who is one of the test developers.
With the addition of an enzyme to saliva samples, the new assay, SalivaDirect, "enables removing expensive and time-consuming RNA extraction now required for RT-PCR testing," Wyllie said in an interview.
Though they have gathered enough data for an FDA submission, the Yale researchers have also launched a study in a collaboration with the National Basketball Association (NBA) and National Basketball Players Association (NBPA) to further validate SalivaDirect. "The big question about any new testing approach is how well it works not only in individuals but also in the larger population," Wyllie said. "By working with the NBA, we've had the opportunity to get more samples to be able to further validate SalivaDirect."
The NBA contacted the Yale research team in mid-May after hearing about its saliva-based test development work. "They were interested in applying our approach to make testing more scalable, affordable, and available to the public," Wyllie said. "Like us, they were thinking about how screening could be done using a less invasive collection method, and they wanted to support our research."
The NBA is well suited to supporting such research because it has an infrastructure for doing drug testing for its professional players, Wyllie said. Further, the league periodically tests its players who must be in close contact without face coverings.
The testing of NBA players is taking on a heightened urgency as the league is scheduled to resume its season on July 30 after temporarily shutting it down in March. Utah Jazz center Rudy Gobert was the first NBA player to test positive for COVID-19.
As it sets to reopen the season, another 16 out of 302 players in the NBA reportedly have tested positive in the league’s first round of coronavirus testing toward the end of June.
Though the saliva tests were not used for that round of testing, a number of the 22 NBA teams have opted into the study with Yale and will participate until they travel to Orlando next week, an NBA spokesperson told 360Dx, though she declined to say specifically how many. The NBA is "hopeful that [its] contributions to this work will help advance research in the interest of broadly accessible and minimally invasive testing," the spokesperson said, but she didn't want to speculate "about what might or might not happen down the line with additional testing," or whether the NBA would use the new saliva assay for testing going forward if it is made available.
In the study with Yale, players' nasopharyngeal swab samples are being sent to Secaucus, New Jersey-based Quest Diagnostics for RT-PCR testing, and saliva samples from the same players are being sent to Yale so that the researchers can compare results for both sample types.
The test's potential for detecting active infections enabled by saliva collection extends beyond sports activities, according to Wyllie. "Our idea has been to develop a protocol that is affordable and as easy as possible to apply on a broad scale," Wyllie said.
Use of the SalivaDirect protocol eliminates the need for RNA extraction and its associated supply chain issues, and it speeds up testing, according to the Yale researchers.
They have been able to obtain performance levels with molecular testing using saliva that are equivalent to tests that use nasopharyngeal swabs, said Chantal Vogels, a researcher at Yale School of Public Health, who helped develop the RNA extraction-free method for SalivaDirect.
The extraction-free method is enabled by treating saliva with an enzyme, proteinase k, prior to introducing the sample for testing by a dual-plex assay on the RT-PCR machine, Vogels said. "We use one dual-plex reaction," she said, "while many other RT-PCR tests use separate reactions for the virus and another to ensure that the sample is adequate for testing."
Using fewer reactions in the overall process drives down the cost of testing, she added.
It also cuts down on the use of reagents. Shortages of reagents, especially RNA extraction kits, are among the reasons for testing shortages during the pandemic. Though testing capacity has increased, the current spike in new cases in the US and elsewhere has emphasized the need for more testing on a mass scale, Wyllie said.
To try to relieve this bottleneck, several labs have been working on assays that add samples directly to the PCR reaction and don't require RNA extraction.
Sample collection for SalivaDirect involves the use of spit-vials that are in plentiful supply and cost about $.15 an item, contributing to the overall affordability of testing and the potential for more testing to be done in symptomatic, pre-symptomatic, and asymptomatic people, Wyllie said.
If the Yale group is successful with its EUA submission, a high-sensitivity SalivaDirect test may be available to the general public later this month at $2 per test, she said. Wyllie and her colleagues have posted the RNA extraction-free saliva protocol to protocols.io. a web application that allows scientists to create, copy, modify, and develop laboratory protocols.
The Yale group has completed the validation of SalivaDirect on clinical samples obtained from Yale New Haven Hospital and anticipates submitting its EUA application this week.
The Yale team doesn't anticipate commercializing the test itself. An EUA would enable self-collection of saliva at home and use of the new protocol by labs that would manage sample collection, testing, and reporting of results, Wyllie said, while she and her colleagues continue to focus on research and development.
In an earlier study, Wyllie and her colleagues compared results from nasopharyngeal swabs and saliva samples from 44 COVID-19 patients with severe disease and found that virus titers were higher in the saliva samples. Saliva showed a much lower false-negative rate than nasopharyngeal swabs, they said.
Additional test developers are looking to provide evidence of the potential for saliva-based SARS-CoV-2 assays. In a recent study published on the MedRxiv preprint server, for example, researchers from Johns Hopkins University, the National Institutes of Health, Emory University, and the University of California, Irvine, compared the performance of a newly developed saliva-based test that targets antibodies against 12 SARS-CoV-2 antigens with that of a serum-based assay targeting the same viral antigens.
The FDA has already granted EUA for a saliva collection kit associated with P23 Labs' TaqPath SARS-CoV-2 PCR test and for an at-home saliva-based collection kit developed by Piscataway, New Jersey-based Rutgers University Cell and DNA Repository (RUCDR) Infinite Biologics.
An RNA extraction-free saliva test, such as the one Yale is developing, could be especially useful where there is a need for broad screening, including of healthy individuals, Scott O’Brien, senior vice president of global marketing and international sales at Carlsbad, California-based GenMark Diagnostics, said in an interview. It's not surprising that the NBA and other sports organizations would be interested in such an approach, he said.
GenMark's ePlex molecular diagnostic tests for SARS-CoV-2 operate with nasopharyngeal swabs, but the firm is also investigating saliva as a sample, he said. Still, nasopharyngeal swabs have proved their clinical utility in testing throughout the pandemic, and any saliva-based approach will have to prove its ability in clinical studies to enable high sensitivity for detection, O'Brien noted.